Biosensing is an important field in industry, and especially in the food industry, where the detection and monitoring of the levels of bio-contaminants, such as bacterial levels in food products, is of critical importance in maintaining modern health standards. In the food industry, the need for constant monitoring of products coming off production lines is particularly important and problematic, since any production fault resulting in bio-contamination of the products must be detected before products are shipped for marketing. This is currently often done by means of testing cultures grown from samples from each production line and batch. However, because of the time taken to grow and tests such cultures, even using modern accelerated culture growth and measurement techniques, a large number of products may have been produced and packed ready for shipping before the contamination is detected, thus leading to considerable loss. There exist technologies, such as surface plasma resonance (SPR) for instance, which enable constant monitoring of the bio-contaminant levels in products, but such technologies are costly—a typical installation costing many tens of thousands of dollars. Because of the large number of different products produced on different production lines, such bio-sensing technologies are often prohibitively expensive for general use in the food industry, and there appear to exist no low cost bio-sensing equipment which can perform cost effective widespread online monitoring of food products.
In recent years, porous Si (hereinafter PSi) has emerged as a promising nanomaterial for bio-sensing applications, and for sensing other targets with nanoscale dimensions. Common PSi-based optical sensors and biosensors consist of thin films of either nano-pores (typically of dimensions less than 20 nm) or meso-pores (typically in the range of 20-100 nm) which thus have cross sectional dimensions much smaller than the optical wavelengths used. The pores are generally randomly generated during production of the thin film device. The operation of these sensors is based on replacing the media in the pores and/or infiltrating with the target analyte, and observing the resulting changes of optical reflectivity. A change in the effective refractive index of the PSi film is manifested as a wavelength shift in the reflectivity spectrum. Only target molecules which penetrate into these nanostructures, can be detected. Indeed, sensing and biosensing of various chemical and biological analytes, such as fluorescent molecules, organophosphates, volatile organic compounds, DNA, and proteins have been successfully demonstrated. Many of these studies employ the method of reflective interferometric Fourier transform spectroscopy (RIFTS) to monitor biological interaction within meso-porous Si thin films.
Such filled or partially filled, randomly located pores can be viewed as simply having a different effective refractive index from that of unfilled pores, because the pores are much smaller than the optical wavelength. Consequently, the random nature of the pores does not result in scatter of the light, but rather in an averaging out of the overall reflected light from the combination of the silicon substrate and the pores, both filled and unfilled. However, this detection scheme is not applicable for targeting large biological or other species, such as those ranging in size of from approximately a few hundreds of nanometers up to several microns and more, including cells, bacteria and viruses. If porous silicon is produced having such larger pores, the substrate becomes a material known as “black silicon”, which appears as such because it strongly scatters light from the random distribution of the large sized pores. Essentially all of the incident light is randomly scattered by the pores and is absorbed in the medium, such that it cannot be used for sensing. Therefore, the prior art porous silicon technology cannot be used for sensing or bio-sensing of larger targets of sizes which are a significant fraction of the wavelength of the light used for the sensing.
There exist alternative methods which monitor changes in the intensity of the reflectivity spectrum upon direct capture of larger cellular targets on top of the biosensor surface, rather than in the pores thereof. However, these types of sensors are limited, since intensity changes of the reflectivity spectrum may arise from unpredictable sources, such as environmental effects and non-specific binding events. In addition, the sensitivity may be low because such surface-binding sensors do not take advantage of the large porous volume.
In recent years attempts have been made to develop new bioassays and biosensors for the rapid detection of bacteria in general, and pathogenic bacteria in particular. However, despite the significant progress in the field, current technologies lack the ability to detect microorganisms in “real time” or outside the laboratory environment
There therefore exists a serious need for a detection method and apparatus which overcomes at least some of the disadvantages of prior art systems and methods, and in particular, is able to perform continuous monitoring of the level of large biological contamination species, such as living cells, pathogenic bacteria, spores and viruses, or of other species of similar size, namely dimensions generally equal to or greater than the wavelength of visible light.
The disclosures of each of the publications mentioned in this section and in other sections of the specification, are hereby incorporated by reference, each in its entirety.